Blood Lactate Analysis In Sports Science: Matrix Considerations

Intact Whole Blood, Plasma or Haemolysed Whole Blood?

Samples for lactate analysis by Analox technology can be plasma, intact whole blood and haemolysed whole blood. The three different matrices give different results, although in practice all three are used. However, to undertake meaningful studies, the reason for the differences should be understood, and values should only be compared to those obtained with the same sample type, i.e. matrix. This is important when interpreting or preparing scientific publications.

The main theoretical difference in the results is due to the distribution of lactate between the different constituents of blood. Lactate is present in the water phase of both the plasma and red cells, but the water content of plasma is higher than the water content of red cells so the distribution of lactate in the blood is not uniform.

Consider the three possible sample matrices for the same specimen volume, as injected into the analyser:
 

For a Plasma sample, the sample will be essentially homogeneous and all the lactate present in plasma water will be measured.

For the same volume of Intact Whole Blood, i.e. where the red cells are complete and have not been ruptured, only the lactate in the plasma water would in theory be measured. The red cells will typically occupy around 50% of the sample volume, but the haematocrit of blood varies according to age, sex and between individuals. The lactate result in intact whole blood could therefore be expected to be around 50% of the plasma value but it is more in fact because partial diffusion of lactate out of the cells occurs during analysis. A range of differences have in fact been observed, varying up to 35%, depending to a large extent on the method of analysis. The average reported shortfall is 25-30% and this is the case with the Analox oxygen-rate approach.

For a similar volume of Haemolysed Whole Blood, i.e. where the red cells have all been ruptured (lysed) and their contents added to the plasma, all the lactate present will be measured, but this will be lower than an equal volume of plasma because:
a) the lactate content in the proportion of red cells is less than the lactate content in the proportion of plasma.
b) there will be some red cell debris contributing to the sample volume.

Again, there are differences between haemolysed whole blood and plasma samples but it is less than the difference between haemolysed and intact whole blood samples. Thus, in summary, different types of the same sample would give:

Plasma result > Haemolysed Whole Blood > Intact Whole Blood

Note also that the relationship between haemolysed whole blood and intact whole blood is not fixed across the whole lactate range as the cellular lactate contribution increases with exercise.

Collection Capillaries:
In the Analox enzymatic oxygen-rate method, whole blood samples are collected in special capillaries treated with chemicals to preserve the lactate values. As the Analox method is an oxygen-measuring technique it is important to ensure that the oxygenation status of the haemoglobin does not cause interference. This is done by converting all the haemoglobin to the 'met-' form using nitrite. Whole blood samples without nitrite may give very distorted results, both high and low, and should NOT be used.

GMRD-053/GMRD-054 capillaries contain heparin (as an anticoagulant), fluoride (as an anti-glycolytic agent) and nitrite. These tubes facilitate analysis after 2-3 minutes, when conversion to met-haemoglobin is complete.

When used with GMRD-090/092/093 lactate reagent kits, these capillaries yield an intact whole blood result. When used with GMRD-100/102/103 lactate reagent kits, which incorporate a lysing agent in the buffer solution, they will produce a haemolysed whole blood result.

Also available are lysing capillary/microtubes (GMRD-070/072) and Analox tubes (GMRD-044/045) which are suitable for delayed analysis and/or postal transport. These systems additionally contain a lysing agent and analyses can be undertaken with either enzyme buffer system.

Glycolysis And Delayed Measurement:
After blood collection, additional lactate is produced from glucose by all the cellular elements including platelets. This is glycolysis. The presence of an anti-glycolytic agent, e.g. fluoride, will retard the process but will not stop it completely.

Thus, in capillaries GMRD-053/054 after 15-20 minutes at room temperature, a rise in the lactate result will begin to be detected. Therefore if an intact whole blood lactate result is required, analyses should be made within this time period.

Glycolysis is also temperature dependent and can be further reduced by lowering temperature. If capillaries are sealed with end-closures and stored in ice/water, i.e. at 0°C, the lactate level should show no significant rise for 8-10 hours. For an intact whole blood lactate result, care must be taken not to freeze the sample. Freezing the sample will rupture the red cells and the blood will be haemolysed to a greater or lesser extent.

If a delayed haemolysed whole blood lactate result is required, the capillaries can be sealed and frozen. This will cause a further slowing of the glycolysis process and hence lactate preservation is enhanced.

In conclusion, it will be seen that when comparing different methods for lactate analysis the situation is much more complicated than it at first seems and no method can be regarded as universally correct in all situations. Different methods give different values and the way the blood is stored and preserved prior to analysis is of profound importance in respect of the values actually obtained. Great care must therefore be taken when comparing data from different methods or samples. The obvious need is to choose a suitable method and sampling technique, and to use them consistently and to state the exact procedure used in any publication.